Diagnostic test for infectious mononucleosis with aldehyde treated equine erythrocytes



4 Claims ABSTRACT OF THE DISCLOSURE Method and composition for the diagnosis of infectious mononucleosis, involving the use of a saline suspension of aldehyde treated equine erythrocytes for the titration of test serums, the presence in such serums of the heterophile antibodies associated with infectious mononucleosis effecting agglutination of the thus treated erythrocytes.

This application is a continuation-in-part of copending application Ser. No. 486,362, filed on Sept. 10, 1965, now abandoned.

This invention relates to a method for the diagnosis of infectious mononucleosis and to a novel composition useful therein; more particularly, the invention relates to an improved test for diagnosing such disease with improved accuracy, rapidity and simplicity.

Infectious mononucleosis is an acute disease most commonly found in patients within the 1625 age group, which is characterized by fever, generalized lymph node enlargement and lymphocytosis with atypical Lymphocytes. Heterophile antibodies, i.e., antibodies having an aflinity for related or identical antigens found in sheep cells, guinea pig tissues and many other unrelated biological substances, are usually present in the blood of mononucleosis patients and, in fact, provide the basis for the commonly employed heterophile antibody test for this disease.

It should be noted that, while heterophile antibodies are found in the sera of 90% of those patients infected with mononucleosis, such antibodies may also be present in the blood of individuals not having the disease. Thus, the antibodies may be produced by the injection of biologicals containing horse serum, particularly in persons who manifest serum sickness following such injections. Alternatively, .they may occur naturally in the blood stream in low titer as so-called 'Forssman antibodies, or native heterophile. It is because of this variety of hetero phile antibodies which may be present in test sera that it has heretofore been necessary to employ relatively complicated multistage serological tests to identify and distinguish the heterophile antibodies characteristic of mononucleosis.

The common serological tests for heterophile antibodies are based upon the fact that such antibodies can be detected by their ability to cause agglutination of sheep erythrocytes. In order to diagnose mononucleosis, making use of such phenomenon, a 3-stage diagnostic test has evolved.

Initially, a slide test is performed, in which the serum suspected to contain heterophile antibodies is titrated with sheep erythrocytes, antibodies present therein agglutinating the erythrocytes.

In the event of a positive agglutination indicating the presence of such antibodies, a second presumptive test nited States Patent 3,426,123 Patented Feb. 4, 1969 is carried out, in which a series of samples of the test serum, in serial dilution, are titrated with the erythrocytes. This test is thus designed to determine the titer of the total heterophile antibody content of the test serum sufficient to produce agglutination of the erythrocytes.

In order to confirm the presumptive test and to distinguish the type of heterophile antibodies present in the test serum, it is usually desirable to conduct a third or differential heterophile test. Such test involves the treatment of two further samples of the original test serum with two antigens, the first of which is capable of adsorbing any Forssman and serum sickness antibodies present in the sample treated, and the second of which is capable of adsorbing any infectious mononucleosis and serum sickness antibodies present in the other sample. Thus, for example, antigen prepared from horse kidneys will adsorb the Forssman and serum sickness antibodies, whereas antigen prepared from beef erythrocytes will adsorb the infectious mononucleosis and serum sickness antibodies. Adsorption of such antibodies, after exposure to either antigen, is indicated by the loss of the serums ability to agglutinate sheep erythrocytes. Hence, after separating the antigen-treated samples, they may be titrated in serial dilution to determine the titers of their residual antibody contents suflicient to produce sheep erythrocyte agglutination.

By subsequently comparing the titer determined in the presumptive test with the titers obtained after adsorption of two further sera samples in the differential test, the type of antibodies present in the test serum may be determined. Employing horse kidney and beef cell antigens,

for example, the heterophile antibody test for infectious mononucleosis is positive if, in the differential test, adsorption of the serum with the horse kidney antigen fails to completely remove the anti-sheep cell agglutinins, whereas adsorption with the beef cell antigen completely removes the anti-sheep agglutinins.

It will thus be seen that relatively complex and timeconsuming serological testing is required to determine the presence of heterophile antibodies characteristic of infectious mononucleosis, and to distinguish such antibodies from those naturally occurring or characteristic of serum sickness. Moreover, it has been found that titration with sheep erythrocytes frequently produces false positive slide test results which necessitate time-consuming further testing, employing the presumptive and differential procedures described above. In one instance, a rate of 9% false positively reacting sera has thus been reported.

I has also been proposed to employ raw horse erythrocytes in place of sheep erythrocytes in the heterophile antibody test for infectious mononucleosis. 2 Such substitution was not, however, found to improve the specificity of the agglutination test and has not, therefore, been advocated. 3

The use of raw sheep erythrocytes as a test reagent for the serological diagnosis of mononucleosis has also been found objectionable because of the necessity for discarding the fresh cells after one weeks storage, and the consequent requirement for frequent preparation of new erythrocyte reagent suspensions. It has been proposed to remedy such problem by preserving the sheep erythrocytes by 1 Davidsoh'n, 1.: Further Studies on Heterophile Antibodies in Serum Sickness, J. Immunolo. 18, 3149 (1930).

P. Beer: The Heterophile Antibodies in Infectious Mon0- nucleosis and after the Injection of Serum. J. Clin. Invest 15. 591-599 (1936) A. M. Barrett, The serological Diagnosis of Glandular Fever (Infections Mononucleosis) A New Technique, J. Hyg. 41, 330-343 (1941).

3 A. M. Barrett: Ibid, p. 334.

4 C. D. Cox: Preservation of Sheep Erythrocytes and Their Use in a. Rapid Plate Titration of Heterophilic Antibodies in lrllfcctious Mononucleosis, J. Lab. & Clin. Med, 48, 293-303 956).

treatment with formalin. Unfortunately, however, the preservation of such cells does not reduce the degree of false positive test results obtained when the preserved sheep erythrocytes are titrated with heterophile agglutinins and does not, therefore, increase the specificity of the heterophile antibody for infectious mononucleosis.

It is, accordingly, among the objects of the present invention to provide a method for diagnosing infectious mononucleosis, which possesses greater specificity for the diagnosis of such disease than the heretofore employed heterophile antibody test employing sheep erythrocytes.

A further object of the invention is to provide such a method which, in view of its improved specificity, is substantially free of false positive indications and does not, therefore, require the high proportion of time-consuming presumptive and differential testing required by heretofore known heterophile antibody serological diagnoses.

Yet a further object of the invention is to provide a composition useful in the improved diagnostic method of the present invention, which composition is stable and may be employed even after long periods of storage.

These and other objects are accomplished in accordance herewith by titrating test sera suspected to contain heterophile antibodies characteristic of infectious mononucleosis with saline suspensions of aldehyde-treated equine, preferably horse, erythrocytes, rather than with raw sheep erythrocytes, as previously employed. The equine erythrocytes may be treated with any suitable water-soluble aldehyde which is compatible with the red blood cells treated, i.e., which will not destroy the same. Aldehydes which have been found so useful include formaldehyde, pyruvaldehyde and glutaraldehyde.

It has been found that the use of aldehyde-treated equine erythrocytes provides a markedly lower rate of false positive results than achieved employing sheet erythocyte test antigens. In one instance, of 300 sera tested, only one showed a false positive agglutination employing a formalized horse erythrocyte serological reagent, corresponding to a rate of only 0.4% false positive results. On the other hand, as indicated above, it has been reported that the titration with sheep erythrocytes has resulted in 9% false positively reacting sera.

Thus, while aldehyde-treated equine erythocytes pro duce strong positive agglutination with sera prepared from patients having infectious mononucleosis, sera having low titers of heterophile antibodies other than those characteristic of infectious mononucleosis do not react with aldehyde-treated equine cells. Slide tests utilizing the aldehydetreated equine cells thus provide more specific positive or negative indications, in many instances eliminating the necessity for further time-consuming serological testing, or at the very least decreasing the degree of presumptive and differential testing subsequently necessary to confirm the presence of antibodies characteristic of infectious mononucleosis. When it is desired to effect such presumptive testing, a series of samples of the test sera, in serial dilution, are titrated with the aldehyde-treated equine cells to determine the titer of the infectious mononucleosis heterophile antibody contents of the test sera sufficient to product agglutination of such cells.

The aldehyde-treated equine erythrocyte compositions employed in the practice of the method hereof are prepared by treating one volume of equine erythrocytes with from 0.2 to one volume of the appropriate aldehyde, preferably formaldehyde. The treatment may be conducted at any suitable temperature, it being preferred to react the reagents at temperatures of from about 4 to 40 C. The thus aldehyde-treated cells are thereafter incorporated in a saline suspension containing from about 2% to 6% by volume of the cells, and desirably, additionally containing a suitable preservative, e.g., sodium azide, phenol, or Merthiolate (a trade name for sodium ethyl mercurithiosalicylate). Reagents thus prepared have been found so stable as to be active after six months or more when stored at temperatures of about 4 C.

The formalized horse erythrocytes or other aldehydetreated equine red cells are thereafter titrated against the test sera, employing a slide test of the type described hereinalbove. Such slide tests provide clear macroscopic evidence of the presence of heterophile antibodies, positively reacting sera displaying coarse agglutination which usually develops within one minute of titration.

The following examples illustrate preferred em bodiments of the method and composition of the present invention; it will, however, be understood that the invention should not be construed as limited to the preferred techniques described therein.

EXAMPLE 1 A 4% saline suspension of formalin-treated horse erythrocytes containing 0.1% sodium azide preservative was prepared. The formalized erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and one-half Alsevers solutin. To 50 ml. of the aforegoing mixture was added 20 ml. of 50% ormalin and the mixture incubated at a temperature of 37 C.

One drop of the saline suspension was added to one drop of each serum to be tested on a fiat glass slide, mixed at room temperature with a wooden applicator stick, rotated for two minutes and read for agglutination within such period, using indirect lighting. A saline control was carried out concurrently with each slide test. Clear or fine granular patterns were regularly viewed in the slide tests employing the saline controls. Positively reacting sera, on the other hand, produced coarse agglutination patterns.

The formalized horse cells thus prepared were utilized in slide tests of 978 different test sera, and the results thus obtained compared with the serological diagnoses obtained employing the slide test-presumptive test-differential test technique described by Davidsohn. Of the 978 tests performed, the results obtained by the use of the simple slide test employing formalized horse erythrocytes differed from those obtained by the differential test procedure in only nine cases, equivalent to a diagnostic accuracy of over 99%. The test results are summarized in Table I below, comparing the positive and negative indications as to the presence of infectious mononucleosis (IM) obtained by the respective tests:

TABLE I.OVERALL TEST RESULTS Davldsohn Differential Test Reaction of Formalized Total Cases Horse Cells 1 Diagnostic accuracy-99%.

A number of the test sera titrated with the formalized horse erythrocytes were also titered against sheep cells to compare the efficacy of slide tests employing the conventional raw sheep cell reagent, with such tests utilizing the formalized horse cell reagent of the present invention. In these latter tests 250 sera obtained from blood donors and patients suffering from a variety of diseases were used as controls. Of these, one serum from a patient with bronchial asthma showed a positive agglutination, while 249 were negative with the formalized horse cells.

117 sera from patients with clinical symptoms suggestive of infectious mononucleosis were also studied. Of these, 28 cases agglutinated both fresh sheep cells and formalized horse cells, 41 sera did not react with either fresh sheep cells or formalized horse cells, and 47 sera reacted positively with the sheep cells and negatively with the formalized horse cells. All of these latter sera were Davidsohn, 1.: Serologic Diagnosis of Infectious Mononucleosis, J.A.M.A., 108, 289-295 (1937).

INFEOTIO US MONONUCLEOSIS have been provided, which promote increased specificity in serological testing for the diagnosis of such disease. Since verious changes may be made in the procedure and compositions described hereinalbove, without departing from the scope of the invention, it will be understood that the preceding description should be interpreted as illustrative and not in a limiting sense.

The embodiments of the invention in which an exclusive privilege or property is claimed are defined as fol- 10 lows:

TABLE II.COMPARISON OF SHEEP AND FORMALIZED HORSE CELL REACTIVITY WITH SERA FROM PATIENTS SUSPECTED OF HAVING Sera Reacting With Non IM Sera Sheep Formallzed Total Cell Titers Davldsohn's Difierentlal Test Sheep Cells Horse Cells Cases 1:7 1:14 1:28 1:56 Pos. (or Neg. tor

IM IM 57 O 3 0 54 l 3 i 69 36 22 10 1 0 69 105 0 105 Control we l 11112313333333:31:13:..."9....

U 1(gne patient had bronchial asthma; one patient had diabetes mellitus; one patient had i Chnvalescent serum. Patient had bronchial asthma.

EXAMPLE 2 A 4% saline suspension of glutaraldehyde (C H O treated horse erythrocytes containing 0.1% sodium azide preservative was prepared. The glutaraldehyde-treated erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and one-half Alsevers. solution. To 50 ml. of the foregoing mixture was added 80 ml. of 25% glutaraldehyde, 16 ml. sodium carbonate to a pH of 7.0, and 50 ml. of 0.15 M phosphate buffer to a pH of 7.2. The mixture was thereafter incubated at 25 C. for 8 hours.

The glutaraldehyde-treated cells thus prepared were utilized in slide tests of 20 different test sera, and the results thus obtained compared with the serologic diagnoses obtained employing the presumptive test-differential test technique described by Davidsohn. Of the 20 tests so performed the results differed from the Davidsohn procedure results in only one case.

EXAMPLE 3 A 4% saline suspension of pymvaldehyde-treated horse erythrocytes containing 0.1% sodium azide preservative was prepared. The pyruvaldehyde erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and onehalf Alsevers solution. To 50 ml. of the foregoing mixture was added 50 m1. of a pyruvaldehyde solution containing 8 ml. of 45% pynu-valdehyde, 17 m1. of a 1% sodium carbonate solution, and ml. of 0.15 M sodium phosphate buffer, the resulting mixture having a pH of 6.5. The mixture was thereafter incubated at a temperature of 25 C.

The pyruvaldehyde-treated horse cells thus prepared were utilized in slide tests of 20' different test sera, and the results thus obtained compared with the serologic diagnoses obtained employing the presumptive test-differential test described by Davidsohn. Of the 20 tests performed, the results obtained by the simple slide test of this example differed in only one case from those obtained by the Davidsohn procedure.

In accordance with the present invention, an improved method for the diagnosis of infectious mononucleosis and a novel composition useful in the practice of such method See footnote 5, column 4.

1. A method for the specific diagnosis of infectious mononucleosis, which comprises titrating a test serum with a saline suspension of aldehyde-treated equine erythrocytes, said erythrocytes having been treated with a water-soluble aldehyde compatible therewith, whereby the presence in said serum of heterophile antibodies associated :with infectious mononucleosis effects agglutination of the thus treated erythocytes.

2. The method of claim 1, said erythrocytes consisting essentially of horse erythrocytes and having been treated with formalin in an amount of from 0.2 to 1 volume of formalin per volume of horse erythrocytes, and in which said suspension contains from 2% to 6% by volume of the thus formalized cells.

3. The method of claim 1, said erythrocytes having been treated with an aldehyde selected from the group consisting of formaldehyde, glutaraldehyde and p-yruvaldehyde.

4. The method of claim 1, in which said suspension additionally contains, a preservative selected from the group consisting of sodium azide, phenol and merthiolate.

References Cited UNITED STATES PATENTS 5/1967 Fulthorpe l6784.5 2/ 1966 Arquilla 167-845 ALBERT T. MEYERS, Primary Examiner.

A. FAGELSON, Assistant Examiner. 

